Establishment of hyperoxic cell culture system for predicting drug-induced liver injury: reducing accumulated lipids in hepatocytes derived from chimeric mice with humanized liver

Abstract

Drug-induced liver injury (DILI) is a major adverse reaction. Species-specific differences between humans and laboratory animals make it difficult to establish evaluation models that can accurately predict DILI in the preclinical phase. Chimeric mice with humanized liver are potential predictive models for understanding DILI. Chimeric mice generated by transplanting human hepatocytes into urokinase-type plasminogen activator/severe combined immunodeficient mice are known to develop fatty liver and show lipid accumulation in isolated hepatocytes. It is speculated that the lipids accumulated in hepatocytes may interfere with DILI assessment. It is known that normal 20% oxygen culture conditions do not meet oxygen demand because oxygen consumption rate is higher than the oxygen supply rate. Therefore, we predicted that hyperoxic cultures could induce hepatocyte function and reduce accumulated lipids. A culture of chimeric mouse hepatocytes in 40% oxygen showed reduced intracellular lipid and triglyceride levels compared to those cultured in 20% oxygen on days 7 and 10. In addition, fatty acid β-oxidation (FAO) activity increased from day 7 under 40% oxygen conditions. On the other hand, FAO activity increased on day 10 under 20% conditions. Microarray and Ingenuity Pathway Analysis showed that lipid metabolism-related pathways were downregulated under 40% oxygen conditions for 7 days, suggesting the involvement of several mechanisms in decreasing lipid levels and increasing FAO. Furthermore, some pathways related to cellular function and maintenance were upregulated under 40% oxygen conditions for 7 days. In conclusion, chimeric mouse hepatocytes cultured under hyperoxic conditions may be useful for predicting DILI.

INTRODUCTION

Drug-induced liver injury (DILI) is a major adverse reaction that causes drug discontinuation and market withdrawal (Hornberg et al., 2014). It is difficult to accurately predict DILI in humans because of the heterogeneity between humans and laboratory animals (Albrecht et al., 2019) Hence, chimeric mice with humanized liver are expected to be useful for predicting DILI in humans. Humanized mice are generated by transplanting human hepatocytes into liver-injured immunodeficient mice (Tateno et al., 2015, Tateno and Kojima, 2020). Since these mice are repopulated with approximately 80% human hepatocytes, their expression profiles of various human-type drug-metabolizing enzymes and transporters are similar to those of humans (Ohtsuki et al., 2014). Recently, chimeric mice have been reported to be useful animal models for predicting DILI related to metabolic activation by drug-metabolizing enzymes (Kakuni et al., 2012; Yamazaki et al., 2016; Sato et al., 2022). Moreover, the usefulness of hepatocytes derived from chimeric mice with humanized liver has also been employed in an in vitro culture system for evaluating drug metabolism and DILI in humans (Yamasaki et al., 2020; Ikeyama et al., 2020).

On the other hand, Tateno et al. (2011) reported the development of fatty liver in chimeric mice with humanized liver generated from urokinase-type plasminogen activator/severe combined immunodeficiency (uPA/SCID) mice (PXB-mice^®, PhoenixBio Co. Ltd., Hiroshima, Japan). Lipid droplets were also observed in hepatocytes isolated from chimeric mouse liver (PXB-cells^®, PhoenixBio Co. Ltd.) (Ikeyama et al., 2020). Therefore, these are useful in vitro and in vivo models to investigate the mechanisms of lipid metabolism in humans. Tateno et al. (2011) found that the administration of human growth hormone (GH), which regulates lipid metabolism via the upregulation of fatty acid metabolic enzymes, attenuated fatty liver in chimeric mice. These findings suggest that regulation by endogenous mouse GH does not act in transplanted human hepatocytes. However, given that DILI was evaluated using chimeric mouse hepatocytes, there is a concern that lipids accumulated in hepatocytes may reduce the predictability of DILI. Therefore, it is necessary to ameliorate lipids accumulated in the hepatocytes.

In this study, we focused on the potential of hyperoxia in cell culture to reduce lipids accumulated in chimeric mouse hepatocytes. The oxygen consumption rate of hepatocytes under normal physiological conditions is 40−90 pmol/s/cm² (Stevens, 1965). However, the oxygen supply across the liquid-gas interface is only 17 pmol/s/cm² due to the low solubility of oxygen in the culture medium under normal 20% oxygen conditions (Sakai et al., 2012). These findings suggest that a normal oxygen culture does not meet the oxygen requirements of hepatocytes. Previously, Liu et al. (2016) showed that increasing the oxygen concentration from 20% to 80% in an incubator decreased intracellular lactate release, suggesting that sufficient oxygen supply activated mitochondrial oxidative phosphorylation. Hypoxia-inducible factor (HIF) is a central regulatory factor for detecting cellular oxygen levels and adapting to microenvironments (Choudhry and Harris, 2018). Activation of HIF-1α and HIF-2α under 1% oxygen culture conditions reduced peroxisome proliferator-activated receptor-γ coactivator-1α-mediated fatty acid β-oxidation (FAO) activity, leading to lipid accumulation in HepG2 (Liu et al., 2014). Based on this background, culture medium under normal 20% oxygen conditions may be anaerobic for hepatocyte cultures, which have a high oxygen demand, and these microenvironments may reduce lipid metabolic capacity in chimeric mouse hepatocytes. We predicted that culturing hepatocytes at oxygen concentrations higher than 20% would serve as an optimal culture environment that mimics in vivo hepatic function, which in turn may attenuate lipids accumulated in chimeric mouse hepatocytes.

It has been reported that the oxygen requirements of human hepatocytes are lower than those of mouse and rat hepatocytes (Stéphenne et al., 2007; Wagner et al., 2011), and the mitochondrial oxidative phosphorylation activity in chimeric mouse hepatocytes is increased when cultured even under 40% oxygen conditions (Ikeyama et al., 2020). However, there have been no studies on the effects of hyperoxic culture on lipids accumulated in chimeric mouse hepatocytes. In the present study, we evaluated the effects of 20% and 40% oxygen culture conditions on the lipids naturally accumulated in chimeric mouse hepatocytes (PXB-cells^®) derived from PXB-mice^® while considering the mechanisms of lipid metabolism, especially FAO activity.

MATERIALS AND METHODS

Isolation of fresh hepatocytes from chimeric mice with humanized liver

Chimeric mice with humanized liver (PXB-mice^®) were generated by the transplantation of commercially available human hepatocytes (1-year-old, boy, Caucasian, BioIVT, Westbury, NY, USA) into cDNA-uPA/SCID mice (cDNA-uPA wild/+/SCID) (Yamasaki et al., 2020). Hepatocytes (PXB-cells^®) were isolated from male chimeric mice (15–22-week old) with blood human albumin (Alb) levels > 10 mg/mL (estimated replacement indexes > 90%) using a two-step collagenase perfusion method, as previously reported (Yamasaki et al., 2020). Blood human albumin concentrations in PXB-mice^® were measured by immunonephelometry in a JEOL BM6050 autoanalyzer (JEOL, Tokyo, Japan) using LX Reagent Eiken Alb II (Eiken Chemical, Tokyo, Japan). The animal study protocol was approved by the Animal Care and Use Committee of PhoenixBio Co., Ltd. All experimental procedures were conducted in accordance with the guidelines provided by the Proper Conduct of Animal Experiments (June 1, 2006; Science Council of Japan).

Cell culture

Isolated hepatocytes suspended in Dulbecco’s modified Eagle medium (DMEM, Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS, Sartorius, Göttingen, Germany) were plated on Corning^® BioCoat^® Collagen I 24-well or 96-well Black/Clear Flat Bottom TC-treated microplates (Corning, Corning, NY, USA) at cell densities of 4.0 × 10⁵ or 7.12 × 10⁴ cells/well, respectively. The cell number and viability were assessed using the trypan blue exclusion test. The seeded hepatocytes were cultured under 20% oxygen (5% CO₂, 95% atmosphere) or 40% oxygen (5% CO₂, 55% N₂) at 37°C for 24 hr. One day after seeding, the medium was changed to dHCGM (DMEM supplemented with 10% FBS, 2% dimethyl sulfoxide [DMSO], 20 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid [HEPES], 44 mM NaHCO₃, 15 μg/mL L-proline, 0.25 μg/mL insulin, 5 × 10⁻⁸ M dexamethasone, 5 ng/mL epidermal growth factor, 0.1 mM L-ascorbic acid 2-phosphate, 100 IU/mL penicillin G, and 100 μg/mL streptomycin), as previously reported (Yamasaki et al., 2020). The hepatocytes were continuously cultured under each oxygen condition for 10 days. The medium was replaced every 2–4 days under 20% oxygen culture and every 1–2 days under 40% oxygen culture, depending on the condition of the cells.

Oil Red O staining

Isolated hepatocytes from chimeric mice were cultured in 24-well plates under 20% or 40% oxygen. On days 3, 7, and 10 after seeding, the cells were washed with phosphate-buffered saline (PBS) and fixed with 10% paraformaldehyde for 15 min. After fixation, hepatocytes were washed with PBS again, placed in 60% isopropanol, and stained with 60% Oil Red O solution (Muto Kagaku Co., Ltd., Tokyo, Japan) at 37°C for 20 min. Hepatocytes were washed again with PBS and observed under a microscope (ECLIPSE Ti2; Nikon Solutions, Tokyo, Japan). After microscopic observation, PBS was removed, and 100% isopropanol was added. The pigment was extracted by shaking for 15 min and then transferred to a 96-well plate. The absorbance at 490 nm was measured using SpectraMax i3x (Molecular Devices, Sunnyvale, CA, USA).

Quantification of triglyceride (TG)

Isolated hepatocytes from chimeric mice were cultured in 24-well plates under 20% or 40% oxygen. TG levels in the hepatocytes and culture supernatants were determined using the enzymatic colorimetric method Cholestest^® TG (Sekisui Medical Co., Ltd., Tokyo, Japan) on days 3, 7, and 10 after seeding, with reference to the manufacturer’s protocol.

Measurement of Fatty acid β-oxidation (FAO) activity

FAO activity was measured using FAOBlue (Funakoshi Co., Ltd., Tokyo, Japan), a fluorescence probe (Uchinomiya et al., 2020). Hepatocytes isolated from chimeric mice were cultured in 96-well plates under 20% or 40% oxygen for 3, 7, and 10 days. After washing with DMEM, the medium was changed to FBS-free dHCGM containing 10 μM FAOBlue dissolved in DMSO (final concentration, 2%) and incubated for 30 min under each oxygen condition. Hepatocytes were washed with PBS, and the fluorescence (excitation, 405 nm; emission, 460 nm) was measured using SpectraMax i3x (Molecular Devices).

Microarray analysis

Total RNA was isolated from fresh hepatocytes (day 0, suspension) in the air (20% oxygen) and cultured hepatocytes (day 7) under each oxygen condition using Direct-zol^TM RNA MicroPrep (Zymo Research Corporation, Irvine, CA, USA). Total RNA was assessed using the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA) and the Agilent RNA 6000 Nano Kit. Total RNA (100 ng per sample) was labeled and hybridized to the Agilent Human Sure Print G3 Human GE V3 8 × 60 K mRNA microarray chip (Agilent Technologies) according to the manufacturer’s protocol. Data were subsequently normalized and analyzed using GeneSpring 14.9.1 software (Agilent Technologies), as per our previously published protocols (Tateno et al., 2011). RNA expression levels were detected with filters, and only the transcripts with detected flags and at least 100% expression value in any one of the two types of cells were accepted as valid. The global gene expression pattern was compared between isolated fresh hepatocytes from chimeric mice (day 0) and cultured hepatocytes (day 7) under each oxygen condition. Consequently, 24,493 valid transcripts were identified. Statistical analysis with GeneSpring was performed using analysis of variance (ANOVA) unpaired T-test and Benjamini-Hochberg FDR to identify differentially expressed genes (DEGs) with fold change (FC) greater than 2 times, and statistical significance was set as p < 0.05.

Ingenuity Pathway Analysis (IPA)^®

The DEGs datasets obtained from microarray analysis were applied to determine the signaling pathways using Ingenuity Pathway Analysis (IPA)^® technology (QIAGEN, Hilden, Germany). IPA was employed to analyze canonical pathways, diseases and bio functions. The IPA regulation Z-score algorithm was used to predict the activation or inhibition of a given pathway. A signaling pathway is proposed to be significantly activated when the absolute Z-score value is greater than 2.0 and the −Log (p-value) is greater than 1.3.

Statistical analysis

All results are expressed as mean ± standard deviation (S.D., n = 3, independent experiments). Statistical significance was analyzed using the Tukey method following two-way ANOVA using the BellCurve for Excel 2.14 (Social Survey Research Information Co., Ltd., Tokyo, Japan). Statistical significance was set at p < 0.05.

RESULTS

Effects of hyperoxic culture on lipids accumulated in chimeric mouse hepatocytes

Sequential changes in lipids accumulated in chimeric mouse hepatocytes under 20% or 40% oxygen conditions were examined (Fig. 1). Lipid droplets were observed in the hepatocytes immediately after isolation from the liver. The number of lipid droplets seemed to decrease in hepatocytes cultured in 40% oxygen compared to those cultured in 20% oxygen from day 7 (Fig. 1A). These findings were confirmed by Oil Red O staining (Fig. 1B). Extraction of the stain with isopropanol showed a significant decrease in lipid content from day 7 of the hepatocytes in 40% oxygen conditions and on day 7 and 10 of the hepatocytes in 40% oxygen conditions compared to those in 20% oxygen conditions (Fig. 1C). TG levels in the hepatocytes, measured using the Cholestest^® TG kit, were also significantly reduced from day 7 under 40% oxygen conditions and on day 7 and 10 of the hepatocytes in 40% oxygen conditions compared to those in 20% oxygen conditions (Fig. 1D). On the other hand, the TG levels in the culture supernatants of the hepatocytes tended to increase, although not significantly, in 40% oxygen compared to 20% oxygen (Fig. 1E).

Fig. 1

Changes of lipids accumulated in chimeric mouse hepatocytes under 20% or 40% oxygen culture conditions. A) Representative microscopic images of pooled hepatocytes isolated from three chimeric mice cultured under each oxygen condition. (× 20 magnification). B) Representative Oil Red O stained images of hepatocytes cultured under each oxygen condition during culture periods. (× 20 magnification). C) Quantitation of dye amounts extracted from hepatocytes after Oil Red O staining. D) The intracellular TG amounts of hepatocyte cultured under each oxygen condition during culture periods. E) The TG amounts in the culture medium under each oxygen condition during culture periods. Two-way ANOVA showed a statistical difference in oxygen conditions and different culture periods. No interaction effect was detected between oxygen conditions and culture periods. All results are expressed as mean ± S.D., (n = 3) *p < 0.05, **p < 0.01, ***p < 0.001 compared to day 0, ^#p < 0.05, ^##p < 0.01 compared to 20% oxygen conditions on the same day.

Effects of hyperoxic culture on FAO in chimeric mouse hepatocytes

We evaluated the effects of the 40% oxygen culture condition on FAO activity in chimeric mouse hepatocytes (Fig. 2). We confirmed that FAO activity could be quantitatively evaluated using FAOBlue, a fluorescent probe, because the commonly employed FAO activator, 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR), enhanced fluorescence counts in a concentration-dependent manner (Smith et al., 2005) (Supplementary Fig. 1). Evaluating the alteration in FAO activity under each oxygen culture condition revealed a significant increase in FAO from day 7 under 40% oxygen conditions. In contrast, under 20% oxygen conditions, the FAO activity increased on day 10. In addition, FAO activity in 40% oxygen conditions was significantly higher than that in 20% oxygen conditions on day 7 (Fig. 2).

Fig. 2

Changes in FAO activity in chimeric mouse hepatocytes under 20% or 40% oxygen culture condition. The hepatocytes isolated from three chimeric mice were cultured in a 96-microwell plate under 20% or 40% oxygen conditions. The FAO activities in hepatocytes were measured under each oxygen condition. Two-way ANOVA showed a statistical difference in oxygen conditions and different culture periods. No interaction effect was detected between oxygen conditions and culture periods. All results are expressed as mean ± S.D., (n = 3) *p < 0.05, **p < 0.01 compared to hepatocytes on day 3 of culture under each oxygen condition, ^##p < 0.01 compared to 20% oxygen conditions on the same day.

Investigation of lipid reduction mechanism by microarray analysis and IPA

Microarray analysis was performed to compare the global gene expressions of hepatocytes cultured for 7 days under each oxygen condition with the expression patterns of freshly isolated hepatocytes (day 0) to comprehensively investigate other possible underlying mechanisms besides FAO activity that reduce lipids accumulated in hepatocytes cultured under 40% oxygen conditions. Fig. 3A summarizes IPA-based canonical pathways. Canonical pathways were classified according to functional categories and demonstrated significantly altered pathways in the 20% and 40% oxygen culture conditions. Significantly inhibited pathways (Z score < −2 and p < 0.05) are indicated with blue highlights and significantly activated pathways (Z score > 2 and p < 0.05) are indicated with orange highlights. As compared to freshly isolated hepatocytes (day 0), many pathways related to lipid metabolism were downregulated after 7 days in 20% oxygen conditions and were even more downregulated after 7 days in 40% oxygen conditions. In contrast, several pathways related to cellular growth/proliferation, cytokine signaling, and cellular function and maintenance were upregulated after 7 days in 40% oxygen conditions, suggesting the activation of cellular functions in 40% oxygen conditions. In addition, nuclear factor erythroid 2-related factor 2 (NRF2)-mediated oxidative stress response was upregulated after culturing under 40% oxygen conditions, and hypoxia signaling in the cardiovascular system was upregulated (albeit insignificantly) after culturing under 20% oxygen conditions, suggesting that 40% oxygen conditions are hyperoxic and 20% oxygen conditions are hypoxic for hepatocytes. Fig. 3B summarizes the results of diseases and bio functions obtained from IPA, which predicts the biological functions affected by fluctuations in gene expression and its influence on activation/inhibition of signaling pathways. In particular, uptake of lipid, uptake of fatty acid, fatty acid metabolism, synthesis of terpenoid, synthesis of lipid, metabolism of terpenoid, and oxidation of lipid were suppressed after culturing under 40% oxygen conditions for 7 days. Metabolism of amino acid and synthesis of amino acid were also suppressed in the 40% oxygen condition. In contrast, transport of molecule, interaction of endothelial cells, secretion of molecule, binding of endothelial cells, interaction of blood cells, phosphorylation of protein, and cell cycle progression were downregulated in the 20% oxygen condition, but not in the 40% oxygen condition. This finding suggests that compared to the 20% oxygen condition, the 40% oxygen condition more likely resembles the in vivo (freshly isolated hepatocytes, day 0) environments. Furthermore, inflammation of organ, cancer of cells, and morbidity or mortality were upregulated in 20% oxygen, but not in 40% oxygen, suggesting hepatocytes might be healthier in 40% oxygen conditions.

Fig. 3

Summary of IPA-based canonical pathways and diseases and bio functions. Changes in mRNA gene clusters were compared between three batches of pooled isolated hepatocytes (day 0) from 2–4 chimeric mice and three batches of pooled cultured hepatocytes (day 7) from 2–4 chimeric mice under each oxygen condition. A) IPA-based Canonical Pathways. B) IPA-based diseases and bio functions. Each number represents the Z-score, a statistical measure of the degree of consistency between the direction of the predicted gene-pathway relationship and the observed gene expression alterations. A Z-score > 2 or < −2 was considered significant, and orange (activated) and blue (inhibited) shades indicated differences in the magnitude of values, respectively. ^anot significant compared to day 0. N/A; data not available.

Figure 4A shows the pathways leading to biological functions from the upstream analysis. Oxidation of lipid, fatty acid metabolism, and uptake of fatty acid, which are of particular interest in this study, were detected by pathway analysis of nine suppressed target genes, including acyl-CoA oxidase 1 (ACOX1), cytochrome P450 4 subfamily A member 11 (CYP4A11), adenosine triphosphate (ATP) binding cassette subfamily D member 3 (ABCD3), pyruvate dehydrogenase kinase 4 (PDK4), acetyl-Coenzyme A acyltransferase 1 (ACAA1), solute carrier family 27 member 1 (SLC27A1), peroxisome proliferator-activated receptor gamma (PPARG), cluster of differentiation 36 (CD36), and acyl-CoA synthetase long-chain family member 1 (ACSL1) by upregulated Enoyl-CoA hydratase and 3-hydroxyacyl CoA dehydrogenase (EHHADH) and downregulated pyroglutamylated RFamide peptide (QRFP), upstream regulators in 40% oxygen conditions (Fig. 4A). As a result, signaling pathways associated with oxidation of lipid, fatty acid metabolism and uptake of fatty acid were inhibited under 40% oxygen conditions (Fig. 4A). In addition, Mesenchyme homeobox 2 (MEOX2), ubiquitin-specific peptidase 7 (USP7), interleukin-33 (IL33), acyl-CoA synthetase short-chain family member 2 (ACSS2), krüppel-like factor 6 (KLF6), nuclear receptor subfamily 2 group F member 2 (NR2F2), and farnesoid X receptor (FXR) ligand-FXR-Retinoic acid-retinoid X receptor (RXR) α, detected as upstream regulators, controlled expression patterns of target genes, including endothelin 1 (EDN1), CD36, perilipin 2 (PLIN2), PPARG, IL15, scavenger receptor class B member 1 (SCARB1), peroxisome proliferator-activated receptor α (PPARA), solute carrier organic anion transporter family member 1B3 (SLCO1B3) and solute carrier family 27 member 5 (SLC27A5), and suppression of uptake of fatty acid was predicted in 40% oxygen conditions (Fig. 4B).

Fig. 4

Estimation of upstream regulators and mechanistic networks for biological functions using IPA. Using IPA-based diseases and bio functions analysis, differences in mRNA expression between hepatocytes cultured on day 0 and day 7 under each oxygen were assessed; these differences are displayed as nodes (genes) and edges (biological relationships). The edge connecting the gene and each function indicates the relationship predicted based on the directional information encoded by the gene expression (blue, inhibited; gray, unpredictable) and the Z-score. A) estimation of mechanistic networks for lipid oxidation, fatty acid metabolism, and fatty acid uptake, B) uptake of fatty acid.

DISCUSSION

Lipid accumulation was observed in freshly isolated hepatocytes, reflecting the pathophysiology of fatty liver in the humanized liver of chimeric mice (Fig. 1A). Lipid and TG quantification in hepatocytes using Oil Red O staining and Cholestest^® TG revealed that the 40% oxygen conditions decreased lipid and TG levels compared to the 20% oxygen conditions (Fig. 1B–D). FAO activity measured by FAOBlue in hepatocytes cultured under 40% oxygen conditions on day 7 was 1.5-fold higher than in those under 20% oxygen conditions (Fig. 2). Therefore, these results suggest that the increase in FAO activity led to a decrease in lipids accumulated in hepatocytes. It has been reported that a decrease in FAO activity by HIF activation under 1% oxygen conditions induces an increase in TG levels in hepatocytes (Liu et al., 2014). In the present microarray analysis and IPA, canonical pathways for hypoxia signaling tended to be upregulated in the hepatocytes cultured in 20% oxygen for 7 days, but were not altered in those cultured in 40% oxygen (Fig. 3A). Considering these findings, the 20% oxygen condition is considered an anaerobic condition for hepatocytes which have high oxygen requirements, and thus it causes an increased tendency of lipid accumulation via HIF. Furthermore, given that IPA-based canonical pathway analysis indicated a downregulation in lipid metabolism-related pathways after 7 days under 40% oxygen conditions compared to 20% oxygen conditions (Fig. 3A), underlying mechanisms other than FAO activation on day 7 could be involved in the decrease in lipid levels accumulated in hepatocytes. When the TG levels in the culture supernatants were measured, they showed an increase under the 40% oxygen condition as compared to the 20% oxygen condition (Fig. 1E). This tendency may involve the suppression of fatty acid uptake from the culture medium or the facilitation of lipid excretion into the culture medium. IPA revealed that the expression of CD36, which is involved in fatty acid uptake, was downregulated under 40% oxygen conditions (Fig. 4A, B). It has been reported that HIF2α upregulates the expression and function of CD36 and then increases intracellular lipid content (Rey et al., 2020). The reduction in lipids under 40% oxygen conditions may be due in part to the suppression of these pathways under aerobic conditions. In the future, it will be necessary to evaluate mRNA, protein, and activity levels earlier than 7 days considering feedback regulation, including genes that are altered in other lipid-related pathways such as oxidation of lipid, fatty acid metabolism, and uptake of fatty acid (Fig. 4A, B).

In contrast, several pathways involved in cellular stress and injury response were upregulated after 7 days under the 40% oxygen condition as opposed to under the 20% oxygen condition (Fig. 3A). In the 10-day culture, the hepatocytes under the 20% oxygen condition could be cultured with a medium change once every 3 days, whereas the hepatocytes under the 40% oxygen condition tended to detach and thus required a medium change every 2 days (data not shown). Since it is hypothesized that the hyperoxic culture may have adversely affected the hepatocytes, we measured the dissolved oxygen concentrations in the culture supernatants to examine whether the 40% oxygen culture reflects physiological conditions (Supplementary Fig. 2). We found that the dissolved oxygen concentration was below detection (approximately 0%) in the 20% oxygen condition, whereas it was 13% in the 40% oxygen condition (Supplementary Fig. 2). Both highly oxygenated blood (pO₂ ≈ 60–70 mmHg ≈ 10–12%) around the portal vein and deoxygenated blood (pO₂ ≈ 25–35 mmHg ≈ 3–5%) around the central vein are known to be present in hepatic tissue (Godoy et al., 2013; Lee-Montiel et al., 2017). It is hypothesized that culturing under 40% oxygen conditions closely resembles physiological oxygen concentrations around the portal vein of a living body while culturing under 20% oxygen supplies lower oxygen to the hepatic tissue. Cellular ATP contents in chimeric mouse hepatocytes cultured under 40% oxygen conditions were similar to those cultured under 20% oxygen conditions. In addition, hAlb secretion under 40% oxygen conditions increased from day 7 onward. Secretion of hAlb under 40% oxygen conditions was higher than that under 20% oxygen conditions on day 3 (Supplementary Fig. 3). In addition, in the IPA-based canonical pathway analysis involving cellular growth, proliferation, and cellular function and maintenance, several pathways were observed to be upregulated after 7 days under 40% oxygen conditions as compared to 7 days under 20% oxygen conditions (Fig. 3A). These data support the hypothesis that a 40% oxygen culture reflects the in vivo hepatic environment, although the effect of medium change also needs to be considered.

Under normal physiological conditions, almost all ATP in hepatocytes is produced in the mitochondria via aerobic respiration. However, it is known that primary hepatocytes are forced to produce most of their ATP through cytosolic glycolysis via an energy shift induced in the conventional 20% oxygen culture due to the abundant glucose in the culture medium as well as the limited oxygen supply (Liu et al., 2014). Liu et al. (2014) reported that primary hepatocytes freshly isolated from rat livers can be made to avoid such energy shifts by replacing sugar resources in the culture medium and providing a hyperoxic culture. In our IPA-based canonical pathway analysis, glucose metabolism-related pathways were observed to be upregulated after 7 days under 20% oxygen culture conditions, but no significant changes were observed under 40% oxygen culture conditions (Fig. 3A). This suggests that the energy shift to glycolysis was avoided and that the hyperoxic culture reflects the biological microenvironments. Glucose consumption and lactate secretion decreased under 40% oxygen conditions compared to 20% oxygen conditions on day 7 (Supplementary Fig. 4). We considered these results may indicate that an energy shift to mitochondrial oxidative phosphorylation occurred, although further studies are needed.

It has been reported that 27% of DILI cases develop fatty liver (Kleiner et al., 2014), and the establishment of an evaluation system to accurately predict DILI is imperative. Currently, it is reported that HepG2, HepaRG, and cryopreserved primary human hepatocytes are employed in predictive models for identification of fatty liver (Donato et al., 2012; Tolosa et al., 2016; Kozyra et al., 2018). PXB-cells^® are fresh hepatocytes isolated from PXB-mice^®. Therefore, hepatocytes isolated from chimeric mice whose lipid levels have been reduced by hyperoxic culture may also be used to evaluate drug-induced fatty liver, and further research on its usefulness is warranted. In addition, if lipid reduction is confirmed in hepatocytes derived from patients with fatty liver under hyperoxic culture, our findings may lead to the development of novel therapies for fatty liver focusing on the mechanisms elaborated in this study.

ACKNOWLEDGMENTS

The authors would like to thank Dr. Yasuyuki Sakai (Professor, Department of Chemical System Engineering, University of Tokyo, Japan) for help with using the FireSting oxygen monitor and Dr. Chihiro Yamasaki and Dr. Mutsumi Inamatsu (PhoenixBio Co., Ltd.) for their technical assistance. Ohtsuki Y. appreciates scholarship support from the Nagai Memorial Research Scholarship from the Pharmaceutical Society of Japan.

Funding

This study was supported by PhoenixBio Co., Ltd., in part by the AMED-MPS project (Grant Number 21be0304201h0005), and by JST SPRING (Grant Number JPMJSP2132).

Conflict of interest

This research received financial support from PhoenixBio Co. Ltd. Yamao M., Kojima Y., and Tateno C. are employees of PhoenixBio Co. Ltd..

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